Annals of Botany 110: 57–69, 2012 doi:10.1093/aob/mcs104, available online at www.aob.oxfordjournals.org

Expression of floral MADS-box genes in Sinofranchetia chinensis (): implications for the nature of the nectar

Jin Hu1,2,3,†, Jian Zhang1,†, Hongyan Shan1 and Zhiduan Chen1,* 1State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China, 2Graduate University of Chinese Academy of Sciences, Beijing 100049, China and 3South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China * For correspondence. E-mail [email protected] †These two authors contributed equally to this paper.

Received: 17 December 2011 Returned for revision: 20 January 2012 Accepted: 26 March 2012 Published electronically: 31 May 2012 Downloaded from

† Background and Aims The perianths of the Lardizabalaceae are diverse. The second-whorl floral organs of Sinofranchetia chinensis (Lardizabalaceae) are nectar leaves. The aim of this study was to explore the nature of this type of floral organ, and to determine its relationship to nectar leaves in other , and to other floral organs in Sinofranchetia chinensis. † Methods Approaches of evolutionary developmental biology were used, including 3′ RACE (rapid amplification http://aob.oxfordjournals.org/ of cDNA ends) for isolating floral MADS-box genes, phylogenetic analysis for reconstructing gene evolutionary history, in situ hybridization and tissue-specific RT-PCR for identifying gene expression patterns and SEM (scan- ning electron microscopy) for observing the epidermal cell morphology of floral organs. † Key Results Fourteen new floral MADS-box genes were isolated from Sinofranchetia chinensis and from two other species of Lardizabalaceae, Holboellia grandiflora and Decaisnea insignis. The phylogenetic analysis of AP3-like genes in Ranunculales showed that three AP3 paralogues from Sinofranchetia chinensis belong to the AP3-I, -II and -III lineages. In situ hybridization results showed that SIchAP3-3 is significantly expressed only in nectar leaves at the late stages of floral development, and SIchAG, a C-class MADS-box gene, is

expressed not only in stamens and carpels, but also in nectar leaves. SEM observation revealed that the at Institute of Botany, CAS on August 24, 2012 adaxial surface of nectar leaves is covered with conical epidermal cells, a hallmark of petaloidy. † Conclusions The gene expression data imply that the nectar leaves in S. chinensis might share a similar genetic regulatory code with other nectar leaves in Ranunculales species. Based on gene expression and morphological evidence, it is considered that the nectar leaves in S. chinensis could be referred to as petals. Furthermore, the study supports the hypothesis that the nectar leaves in some Ranunculales species might be derived from stamens.

Key words: Nectar leaves, perianth, petals, Ranunculales, Lardizabalaceae, Sinofranchetia chinensis, MADS-box, expression pattern, evolutionary developmental biology.

INTRODUCTION Ranunculales, the earliest-diverging lineage in , displays extreme diversity in perianth morphology and has The reproductive organs of most angiosperms are enclosed by a received increasing attention from evolutionary-developmental sterile outer structure, which is usually called the perianth. The biologists (e.g. Albert et al., 1998; Kramer et al., 1998, 2003, perianth, as a remarkable novelty in angiosperms, generally 2007; Kramer and Irish, 1999, 2000; Theissen et al., 2002; contains two whorls and shows great morphological diversity. Rasmussen et al., 2009; Kramer and Hodges, 2010; Sharma The perianth shape ranges in different lineages from undifferen- et al., 2011). This order is composed of seven families tiated to bipartite (i.e. differentiated into and petals; according to recent molecular phylogenetic studies: Cronquist, 1988; Takhtajan, 1997; Zanis et al., 2003; Endress Ranunculaceae, Berberidaceae, Menispermaceae, Lardiza- and Matthews, 2006; Ronse De Craene, 2008). The various balaceae, Circaeasteraceae, Papaveraceae and Eupteleaceae morphologies of perianths in different angiosperm lineages (Angiosperm Phylogeny Group III, 2009; Wang et al., 2009). have led to the suggestion that perianths could have different The majority of genera in the Ranunculaceae, Berberidaceae, origins (Endress, 1994, 2006). Particularly, petals (the inner Menispermaceae, Lardizabalaceae and Papaveraceae have part of the perianth) have been thought to have evolved several bipartite perianths, whereas there is no clear differentiation of times independently from sterile stamens (andropetals) or the perianth in Circaeasteraceae, and the flowers lack perianths bracts (bracteopetals) during angiosperm evolution (e.g. in Eupteleaceae (Qin, 1997; Damerval and Nadot, 2007; Eames, 1961; Weberling, 1989; Friis and Endress, 1990; Rasmussen et al., 2009; Takhtajan, 2009; Wang et al., 2009; Takhtajan, 1991; Endress, 1994, 2001). However, the molecular Zhang and Ren, 2011). Diverse perianth architectures are evolutionary mechanisms responsible for the different origins of present in Ranunculales, including petaloid sepals, floral nec- petals remain unclear. tariferous organs and spurs (Qin, 1997; Damerval and Nadot, # The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 58 Hu et al. — Expression of floral MADS-box genes in Sinofranchetia

2007; Rasmussen et al., 2009; Takhtajan, 2009; Wang et al., relatively well for most core eudicot flowers with well- 2009; Zhang et al., 2009; Zhang and Ren, 2011). The floral differentiated sepals and petals, but not for most species of nectariferous organs are also called nectar leaves, which are basal eudicots and basal angiosperms with less derived peri- nectar-bearing and formed between the perianth and androe- anth architecture (Soltis et al., 2007). Accordingly, some cium (Janchen, 1949). In Ranunculales, nectar leaves are modified ABCE models have been put forward, such as the found in some species of Ranunculaceae, Berberidaceae, ‘sliding boundary’ model and ‘fading borders’ model (Soltis Menispermaceae and Lardizabalaceae (Erbar et al., 1998; et al., 2007). The ‘sliding boundary’ model suggests that the Ronse De Craene, 2010). Furthermore, nectar leaves in differ- boundary of B-class gene expression can slide across the ent species of Ranunculales display different morphologies: developing flower from its pre-existing location to the outer some are small and greenish, such as in Sinofranchetia perianth whorl (e.g. Kanno et al., 2003; Kramer et al., 2003; (Lardizabalaceae); some are large and petaloid, such as in Ochiai et al., 2004; Kramer and Jaramillo, 2005; Hintz Ranunculus (Ranunculaceae); some are converted to long et al., 2006; Kramer and Zimmer, 2006; Ronse De Craene, spurs, such as in Aquilegia (Ranunculaceae) (Leppik, 1988; 2007). In addition, the labile petal/stamen boundary in the Weberling, 1989; Zhang et al., 2009). The diversification of flower has been suggested to correspond to the sliding of the

nectar leaves in different species of Ranunculales is also A–C boundary (Goto et al., 2001; Theissen, 2001a; Kim Downloaded from reflected in floral morphogenesis: some types of nectar leaves et al., 2005; Chanderbali et al., 2006, 2009; Xu et al., 2006; share common primordia with stamens, such as in Dubois et al. , 2010). The ‘fading borders’ model suggests Berberidaceae and Holboellia (Lardizabalaceae); some that the gradual transitions in floral organ morphology are develop from individual primordia distinguished from that of due to the gradient in the expression levels of floral organ iden- stamens, such as in Sinofranchetia (Lardizabalaceae) (Zhang tity genes (Buzgo et al., 2004; Kim et al., 2005; Soltis et al., et al., 2009; Ronse De Craene, 2010; Zhang and Ren, 2011). 2006; Soltis et al., 2007). http://aob.oxfordjournals.org/ In addition, the nectar leaves have ever been referred to as Both the ABCE model and its derived models emphasize ‘petals’, especially in some species of Ranunculaceae and the importance of B-class MADS-box genes for petal identity Berberidaceae, because these nectar leaves are colourful, specification in core eudicots and for the development of sterile and positioned in the second whorl of the bipartite peri- petal-like structures in basal eudicots and basal angiosperms anth, fitting the broader definition of petals (e.g. Cronquist, (Weigel and Meyerowitz, 1994; Albert et al., 1998; Kramer 1981; Kramer et al., 2007; Kramer and Hodges, 2010). et al., 1998, 2003, 2007; Kramer and Irish, 2000; Theissen However, some other studies considered that the term ‘petals’ et al., 2002; Lamb and Irish, 2003; Aoki et al., 2004; for these organs is mainly related to the function of display Kim et al., 2004; Kramer and Jaramillo, 2005; Zahn et al., at Institute of Botany, CAS on August 24, 2012 rather than the morphological concept (Leppik, 1988; 2005b; Rasmussen et al., 2009; Sharma et al., 2011). Weberling, 1989). Furthermore, some previous studies of the Phylogenetic studies have indicated that two major gene dupli- species of Ranunculaceae and Berberidaceae suggested that cation events occurred during the evolution of B-class genes, the nectar leaves might be derived from the sterilization of one before the origin of angiosperms giving rise to the AP3 stamens (Endress, 1995; Ronse De Craene, 2010). and PI lineages, and the other before the divergence of core In the past two decades, great advances have been made in eudicots leading to the euAP3 and TM6 lineages (Kramer evolutionary developmental biology studies of angiosperm et al., 1998, 2006; Kramer and Irish, 2000; Aoki et al., flowers. New models and theories have been proposed based 2004; Kim et al., 2004; Stellari et al., 2004). These gene on the evolution, expression and functional analyses of duplication events and subsequent functional diversification genes involved in floral development, besides floral morph- have resulted in modifications of floral organ identity ology and morphogenesis (Albert et al., 1998; Baum and programmes in different groups (Kramer et al., 1998, Whitlock, 1999; Kramer and Irish, 1999, 2000; Irish, 2003; 2003; Rasmussen et al., 2009; Specht and Bartlett, 2009). It Kramer et al., 2003; Soltis et al., 2004). These models and the- was also found that two recent gene duplication events had ories promote our understanding of the molecular mechanisms occurred during the evolution of AP3-like genes in for the origin and evolution of angiosperm flowers. Among Ranunculales, giving rise to three AP3 lineages (AP3-I, -II them, the best-known one is the floral ABCE model, which and -III), which in turn enable gene subfunctionalization was proposed from genetic analyses of two core eudicot (Kramer et al., 2003; Rasmussen et al., 2009; Sharma et al., species, Arabidopsis thaliana and Antirrhinum majus. In this 2011). The recent studies in Ranunculales have suggested model, the identity of floral organs is determined by the that the diversification of AP3-like genes is responsible for combinations of four classes of genes: A + E class genes are petaloidy diversity (e.g. Rasmussen et al., 2009; Specht and responsible for the specification of sepals, A + B + E for Bartlett, 2009). In particular, the genes from the AP3-III petals, B + C + E for stamens and C + E for carpels (Coen lineage are found to be petal-specific in the Ranunculaceae and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; and Berberidaceae (Kramer et al., 2003; Rasmussen et al., Colombo et al., 1995; Ma and dePamphilis, 2000; Pelaz 2009; Sharma et al., 2011). Therefore, AP3-like genes are et al., 2000; Theissen, 2001b). Most of the A-, B-, C- and good candidates for studying the molecular mechanisms regu- E-class genes are MIKCC-type MADS-box genes and they lating petal or petal-like structure specification across different belong to the AP1/FUL (A-class), AP3/PI (B-class), AG species. (C-class) and SEP (E-class) MADS-box gene subfamilies, re- In this study, as an initial step towards understanding the spectively (Litt and Irish, 2003; Kramer et al., 2004; Irish and development of nectar leaves in Lardizabalaceae at the molecu- Litt, 2005; Zahn et al., 2005a, b, 2006; Kramer and Zimmer, lar level, we identified the floral MADS-box genes in 2006; Shan et al., 2007, 2009). The ABCE model works Sinofranchetia chinensis and especially studied the evolution Hu et al. — Expression of floral MADS-box genes in Sinofranchetia 59 of the B-class MADS-box genes in Ranunculales. Furthermore, Meixian County, Shaanxi Province, China. They were treated we investigated the expression patterns of the floral MADS-box in one of three ways: fixed in FAA (formalin to acetic acid genes and observed the epidermal cell morphology of floral to 50% alcohol in the ratio of 5 : 6 : 89) for morphological ob- organs in S. chinensis. By integrating molecular, developmental servation; stored in liquid nitrogen for RNA isolation; or fixed and morphological data, we hope to explore the nature of the in PFA (4 % paraformaldehyde) and embedded in Paraplast nectar leaves in S. chinensis, and reveal its relationship to (Sigma, St Louis, MO, USA) for in situ hybridization. Plant nectar leaves in other Ranunculales species, and to other materials of Holboellia grandiflora and Decaisnea insignis floral organs in S. chinensis. were also collected for RNA extraction.

MATERIALS AND METHODS Gene cloning Plant materials Total RNA of floral buds and young flowers was extracted Sinofranchetia chinensis is a liana with unisexual flowers. The using Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA). functionally unisexual flowers are bisexual in organization at Poly(A) mRNA was purified from total RNA using the Downloaded from the floral bud stages, and the unisexuality is found in mature Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany). flowers (Zhang et al., 2009). A Sinofranchetia flower has First-strand cDNA was synthesized using SuperScriptTM III petaloid sepals with a purple margin in the first whorl, greenish Reverse Transcriptase (Invitrogen). We performed 3′ RACE nectar leaves in the second whorl, stamens or sterile stami- (rapid amplification of cDNA ends) PCR with the degenerate nodes in the third whorl, and rudimentary carpels or carpels primers and the adapter primer AP (5′-CCGGATCCTCTACA ′ in the forth whorl (Figs 1 and 6A). Floral buds and mature GCGGCCGC-3 ). To clone B-class MADS-box genes, hemi- http://aob.oxfordjournals.org/ flowers of S. chinensis at different developmental stages nested PCR assay was carried out with the B-class gene- were collected from Taibai Mountain (1200–1500 m a.s.l.), specific degenerate primer B1 and the adapter primer AP.

A B C at Institute of Botany, CAS on August 24, 2012

D E

F IG. 1. Morphology of Sinofranchetia chinensis flowers: (A) plant with inflorescences; (B) female inflorescence; (C) male inflorescence; (D) female flower; (E) male flower. Red asterisks indicate sepals; red arrows indicate nectar leaves. Scale bars ¼ 2 mm. 60 Hu et al. — Expression of floral MADS-box genes in Sinofranchetia

The PCR reaction was heated at 94 8C for 4 min, followed by invariable sites and gamma distribution parameter values ten cycles of 94 8C for 30 s, 48 8C for 30 s and 72 8C for for the GTR + I + G model were optimized by using 1 min, and then 25 cycles of 94 8C for 30 s, 52 8C for 30 s MODELTEST. Each ML analysis used a BIONJ as a start- and 72 8C for 1 min, and finally extended at 72 8C for ing point (Gascuel, 1997). The support value for each tree 10 min. A second B-class gene-specific degenerate primer branch was based on the best ML tree filtered through 1000 B2 and AP were then used to amplify the PCR products bootstrap replicates in PhyML (Felsenstein, 1985). obtained in the first step. The PCR was performed with 35 cycles, and the annealing temperature was set at 52 8C. The amplified fragments over 800 bp were purified and cloned In situ hybridization into the pGEM-T easy vector (Promega, Madison, WI, The floral buds of S. chinensis at various developmental USA). The plasmid DNA was isolated by alkaline lysis stages were used for in situ hybridization. The gene-specific precipitation (Sambrook et al., 1989). The positive primers were designed for amplifying the partial CDS clones were identified by restriction enzyme analysis of the (coding sequences) and 3′ UTR (untranslated regions) of plasmids, and at least three independent clones were Sinofranchetia floral MADS-box genes to make RNA probes sequenced for each identified locus using forward primer T7 (Supplementary Data Table S2). The sense and antisense Downloaded from (5′-TAATACGACTCACTATAGGG-3′) and reverse primer ′ ′ digoxigenin-labelled RNA probes for in situ hybridization SP6 (5 -ATTTAGGTGACACTATAG-3 ). In a similar way, were synthesized using a DIG northern starter kit (Roche other floral MADS-box genes were cloned. Only nucleotide Diagnostics, Mannheim, Germany). Embedding of plant mate- sequences with Phred quality scores .20 were used for rials, pretreatment, hybridization and washing of the sections further analysis. The degenerate primers for B-, C- and were performed as previously described (Li et al., 2005; http://aob.oxfordjournals.org/ E-class gene amplification and the number of sequenced Shan et al., 2006) with slight modifications. clones for each gene are listed in Supplementary Data Table S1.

Sequence retrieval and alignment Tissue-specific RT-PCR Aside from all the floral MADS-box genes cloned from The expression patterns of AP3, PI and AG homologues in the S. chinensis, H. grandiflora and D. insignis, we also obtained mature flowers of S. chinensis were investigated by using tissue- homologous sequences from other species using BLAST specific RT-PCR. The RNA used in RT-PCR was extracted from sepals, petals, staminodes and carpels of mature female

against the publicly available databases, including NCBI at Institute of Botany, CAS on August 24, 2012 (http://www.ncbi.nlm.nih.gov) and the TIGR plant transcript flowers, and sepals, petals and stamens of mature male flowers assembly database (http://plantta.jcvi.org/). The full-length and from young leaves. The rudimentary carpels in the male amino acid sequences for phylogenetic analyses of A-, B-, flowers are too small to be collected, so they were not included C- and E-class MADS-box genes (referred to as global ana- in the analysis. The extraction of total RNA, purification of poly lysis hereafter), AP3-like genes in Ranunculales (referred to (A) mRNA, and synthesis of the first-strand cDNA were per- as AP3 analysis hereafter) and PI-like genes in Ranunculales formed according to the methods described above. The (referred to as PI analysis hereafter) were aligned with amounts of templates were normalized using the control gene ClustalX 1.83 using the default parameters (Thompson et al., ACTIN. Gene-specific forward and reverse primers were used 1997). Alignments were adjusted manually using GeneDoc to detect gene expression (Supplementary Data Table S2). The (Nicholas and Nicholas, 1997). The corresponding DNA PCR thermocycling conditions used were: initial denaturation matrices were generated by aa2dna (https://homes.bio. at 94 8C for 3 min, 30 cycles of 94 8C for 30 s, 55–60 8C(de- psu.edu/people/faculty/nei/software.htm) using the well- pending on the melting temperature of primer pairs) for 30 s, aligned amino acid matrices. In addition, we used ClustalX and 72 8C for 1 min, and a final extension at 72 8Cfor 1.83 to estimate the column scores of the amino acid 10 min. The PCR products were fractionated in 1 % agarose matrices for the global analysis, AP3 analysis and PI analysis, gels and digitally photographed. We repeated the RT-PCR respectively, and the residues with higher-than-12 quality experiments three times independently. scores were kept in the alignment (Thompson et al., 1997; Zahn et al., 2005a; Shan et al., 2007). Based on these residues, Scanning electron microscopy we generated the corresponding nucleotide matrices for further phylogenetic analyses, and the length of the dataset for the The characteristics of the epidermal cells are an important global analysis was 588 bp, 606 bp for the AP3 analysis and criterion for identifying morphological equivalents or homolo- 612 bp for the PI analysis. gues among different floral organs (Endress, 1994; Krizek et al., 2000; Pelaz et al., 2000; Jaramillo and Kramer, 2004; Geuten et al., 2006). We therefore performed SEM (scanning Phylogenetic analysis electron microscopy) analysis of epidermal cell shapes for the Phylogenetic analyses were performed for each DNA matrix , nectar , sterile staminode/stamen and carpel/ using the maximum likelihood (ML) method in PhyML rudimentary carpel in the female and male flowers of version 2.4.4 (Guindon and Gascuel, 2003). The best fit S. chinensis. Young flowers were collected at 7-d intervals model of nucleotide evolution for the DNA matrices was and immediately fixed with FAA. Subsequently, the materials GTR + I + G, which was chosen by running MODELTEST were dehydrated in an alcohol–isoamyl acetate series, treated version 3.06 (Posada and Crandall, 1998). The proportion of by critical point drying in CO2, mounted, and then coated with Hu et al. — Expression of floral MADS-box genes in Sinofranchetia 61 gold. Observations of the epidermal cell morphology of differ- ent floral organs were performed using a Hitachi S-800 scan- ning electron microscope. 100 Petunia h pMADS12 100 Petunia h EBP5 98 Antirrhinum m DEFH49 100 Arabidopsis t SEP1 RESULTS Arabidopsis t SEP2 87 100 Petunia h FBP23 Petunia h FBP9 94 Arabidopsis t SEP4 A-, B-, C- and E-class MADS-box genes in S. chinensis Petunia h FBP4 84 Akebia t AktSEP1-1 Euptelea p EUplSEP1 Six floral MADS-box genes were isolated from S. chinensis, 82 Akebia t AktSEP1-2 100 Oryza s OsMADS1 four from H. grandiflora and four from D. insignis. Through Oryza s OsMADS5 E 82 Asparagus o AOMADS3 BLAST against NCBI databases, these genes were preliminar- 62 Chrysanthemum m CDM44 86 Petunia h FBP2 100 ily grouped into B-, C- and E-class MADS-box genes, and 94 Antirrhinum m DEFH72 100 Antirrhinum m DEFH200 these classifications were further supported by ML analysis Arabidopsis t SEP3 99 100 Akebia t AktAEP3 (Fig. 2). The studied species, gene name and accession 89 Sinofranchetia c SlchSEP3 Euptelea p EUplSEP3 number for 14 newly isolated genes in this study, two previ- 80 53 Pachysandra t PAteSEP3-2 83 Pachysandra t PAteSEP3-1 Downloaded from ously published genes from S. chinensis (Shan et al., 2007) 87 Asparagus o AOMADS2 51 Asparagus o AOM1 and 92 representative floral MADS-box genes from other 100 Oryza s OsMADS7 Oryza s OsMADS8 species downloaded from databases are listed in 58 Petunia h FBP29 68 Chrysanthemum m CDM41 Supplementary Data Table S3. All floral MADS-box genes 100 Arabidopsis t AGL79 100 Petunia h FBP26 analysed here formed four distinct well-supported clades in Petunia h PFG 59 Chrysanthemum m CDM8 the ML tree, corresponding to the AP1/FUL (A-class), AP3/ 91 Antirrhinum m SQUA http://aob.oxfordjournals.org/ 91 Chrysanthemum m CDM111 PI AG SEP 100 Arabidopsis t AP1 (B-class), (C-class) and (E-class) subfamilies of Arabidopsis t CAL MADS-box genes (Fig. 2). Furthermore, the relationships Arabidopsis t FUL 92 100 Pachysandra t PAteFL1 88 Pachysandra t PAteFL3 A among these genes are largely consistent with the species Pachysandra t PAteFL2 Akebia t AktFL1 phylogeny (Fig. 2). Accordingly, the eight floral MADS-box 99 Decaisnea i DEinFL1 61 Sinofranchetia c SlchFL1 genes from S. chinensis were classified as two FUL-like 100 61 Akebia t AktFL2 62 Sinofranchetia c SlchFL2 genes (SIchFL1 and SIchFL2), three AP3-like genes 96 Decaisnea i DEinFL2 75 Euptelea p EUplFL2 (SIchAP3-1, SIchAP3-2 and SIchAP3-3), one PI-like gene 95 Euptelea p EUplFL1 100 Oryza s OsMADS15 (SIchPI), one AG-like gene (SIchAG) and one SEP3-like Oryza s OsMADS14 98 Petunia h FBP6 at Institute of Botany, CAS on August 24, 2012 gene (SIchSEP3); the four floral MADS-box genes from 66 Antirrhinum m PLE 100 Arabidopsis t SHP2 H. grandiflora were one AP3-like gene (HOgrAP3), one Arabidopsis t SHP1 71 Antirrhinum m PAR 59 PI-like gene (HOgrPI) and two AG-like genes (HOgrAG1 93 Petunia h pMADS3 76 Chrysanthemum m CDM37 and HOgrAG2); and the four floral MADS-box genes from Arabidopsis t AG 99 Akebia t AktAG1 D. insignis were two AP3-like genes (DEinAP3-1 and Holboellia g HOgrAG2 100 Decaisnea i DEinAG C 55 DEinAP3-2), one PI-like gene (DEinPI) and one AG-like 65 Sinofranchetia c SlchAG Holboellia g HOgrAG1 gene (DEinAG ) (Fig. 2). The phylogenetic analysis indicated 93 Euptelea p EUplAG1 Euptelea p EUplAG2 that the following paralogous gene pairs might be the result 50 Pachysandra t PAteAG 100 70 Oryza s OsMADS13 of gene duplication events that took place before the diver- Oryza s OsMADS3 100 Petunia h FBP7 gence of the Lardizabalaceae (Fig. 2): SIchFL1 and Petunia h FBP11 Arabidopsis t STK SIchFL2; SIchAP3-1, SIchAP3-2 and SIchAP3-3; HOgrAG1 86 Akebia t AktAG2 Agapanthus p ApMADS2 100 Chrysanthemum m CDM19 and HOgrAG2; and DEinAP3-1 and DEinAP3-2. 96 Chrysanthemum m CDM115 93 Arabidopsis t AP3 98 Petunia h phDEF Antirrhinum m DEF Petunia h phTM6 Sequence and phylogenetic analysis of B-class MADS-box genes 100 Pachysandra t PAteAP3-1 94 Pachysandra t PAteAP3-2 in Ranunculales 99 Akebia t AktAP3-3 99 Akebia t AktAP3-2 98 Holboellia g HOgrAP3 Our phylogenetic analysis of AP3-like genes in Ranunculales 99 Sinofranchetia c SlchAP3-1 B 51 Decaisnea i DEinAP3-2 100 61 showed that the three AP3-like genes from S. chinensis were 98 Akebia t AktAP3-1 Sinofranchetia c SlchAP3-2 grouped into three paralogous AP3 lineages of Ranunculales Decaisnea i DEinAP3-1 99 Sinofranchetia c SlchAP3-3 Euptelea p EUplAP3-2 (Fig. 3 and Supplementary Data Table S4). SIchAP3-1 belongs Euptelea p EUplAP3-1 Oryza s SPW1 to the AP3-I lineage, SIchAP3-2 to the AP3-II lineage, and 62 Chrysanthemum m CDM86 97 Petunia h FBP1 SIchAP3-3 to the AP3-III lineage. Furthermore, SIchAP3-1 89 Petunia h pMADS2 Arabidopsis t Pl and SIchAP3-2 protein sequences show 67 % identity; 100 Holboellia g HOgrPl 90 65 Akebia t AktPl SIchAP3-2 and SIchAP3-3 share 62 % identity; and 99 Sinofranchetia c SlchPl 67 99 Decaisnea i DEinPl SIchAP3-1 and SIchAP3-3 share 56 % identity. The highly con- Euptelea p EUplPl 51 Pachysandra t PAtePl served MADS domain and K domain, as well as PI-derived Oryza s OsMADS4 motif and paleoAP3 motif, were found in the three SIchAP3 Arabidopsis t SVP proteins from multiple sequence alignment with other 0·1 AP3-like proteins of Ranunculales; however, the SIchAP3-2 F IG. 2. Phylogenetic relationships of 108 representative floral MADS-box protein lacks 29 amino acids in the K domain (Supplementary genes. Bootstrap values (.50 %) are shown above the branches. Genes Data Fig. S1). At least four families (Ranunculaceae, obtained in this study are indicated with dots. 62 Hu et al. — Expression of floral MADS-box genes in Sinofranchetia

100 Clematis i CliAP3-1 63 100 Clematis a ClaAP3-1 Anemone n AnnAP3-1 67 Hepatica h HeheAP3-1 56 Trautvetteria c TrcAP3 100 Ranunculus b RbAP3-1 97 Ranunculus f RfAP3-1 Helleborus h HhAP3-1 Caltha p CapAP3-1 Trollius l TllAP3-1 100 Actaea a AcaAP3-1 97 Cimicifuga r CirAP3-1 100 Delphinium e DleAP3-1 Aconitum s AcsAP3-1 100 Thalictrum d ThdAP3-1 67 100 Thalictrum t ThtAP3-1 AP3-I Aquilegia a AqaAP3-1 Hydrastis c HycAP3-1 100 Jeffersonia d JdAP3-1 Epimedium g EpgAP3-3 100 Menispermum d MndAP3-1 Cocculus t CctAP3-1 80 Holboellia g HOgrAP3 100 Holboellia g HbcAP3-1 70 Downloaded from 90 Akebia q AkqAP3-1 Sinofranchetia c SlchAP3-1 Decaisnea i DEinAP3-1 96 Eschscholzia c EcDEF1 92 79 Sanguinaria c ScAP3 Papaver n PnAP3-2 Dicentra e DeAP3 100 Ranunculus f RfAP3-2 Ranunculus b RbAP3-2 72 http://aob.oxfordjournals.org/ 78 Anemone n AnnAP3-2 100 100 Clematis a ClaAP3-2 Clematis i CliAP3-2 Hepatica h HeheAP3-2 Caltha p CapAP3-2 Nigella s NgsAP3-2 Adonis v AdvAP3-2 100 Aconitum s AcsAP3-2 Delphinium e DleAP3-2 100 Actaea a AcaAP3-2 AP3-II 56 Cimicifuga r CirAP3-2 Helleborus h HhAP3-2 97 Aquilegia a AqaAP3-2 99 Thalictrum d ThdAP3-2b 98 100 Thalictrum t ThtAP3-2b at Institute of Botany, CAS on August 24, 2012 100 Thalictrum d ThdAP3-2a 56 Thalictrum t ThtAP3-2a Trollius l TllAP3-2 51 Xanthorhiza s XsAP3-2 Berberis g BgAP3-2 99 Menispermum d MndAP3-2 Cocculus t CctAP3-2 100 Akebia q AkqAP3-2 97 Holboellia c HbcAP3-2 Sinofranchetia c SlchAP3-2 57 Decaisnea i DEinAP3-2 100 Delphinium e DleAP3-3 63 Aconitum s AcsAP3-3 Nigella s NgsAP3-3 Aquilegia a AqaAP3-3 Cimicifuga r CirAP3-3 100 Anemone n AnnAP3-3 Clematis a ClaAP3-3 100 Helleborus h HhAP3-3a Helleborus h HhAP3-3b Xanthorhiza s XsAP3-3 83 100 Adonis v AdvAP3-3 AP3-III 97 Trollius l TllAP3-3 Ranunculus f RfAP3-3 99 Berberis g BgAP3-1 Epimedium g EpgAP3-1 100 Cocculus t CctAP3-3 Menispermum d MndAP3-3 100 Kingdonia u KiuAP3a Kingdonia u KiuAP3b SlchAP3-3 100 Papaver n PnAP3-1 Eschccholzia c EcDEF2 Euptelea p EupAP3 100 Pachysandra p PpAP3-2 99 74 Pachysandra p PpAP3-2 72 Pachysandra p PpAP3-3 57 Trochodendron a TraAP3 Platanus o PloAP3-1 Nelumbo n NnAP3 76 100 Meliosma d MdAP3-3 Meliosma d MdAP3-2 Meliosma d MdAP3-1

0·1

F IG. 3. Maximum-likelihood tree of AP3-like genes in Ranunculales. Bootstrap values (.50 %) are shown above the branches. In Ranunculales, the inferred two major, successive gene duplication events are highlighted by stars, and the inferred small-scale gene duplication events are indicated with dots. Hu et al. — Expression of floral MADS-box genes in Sinofranchetia 63

Berberidaceae, Menispermaceae and Lardizabalaceae) of developing stamens, but the expression level in the carpels Ranunculales have all three paralogues of AP3-like genes, but reduces gradually during floral development (Fig. 4B). At the relationships among these three AP3 lineages in the late stages, SIchAP3-1 expression is mainly restricted to Ranunculales are still uncertain (Fig. 3). the nectar leaves and stamens (Fig. 4C). The expression In this study, we have added three new PI homologues from pattern of SIchAP3-2 is mostly like that of SIchAP3-1, but Lardizabalaceae to the phylogenetic analysis of PI-like genes weak expression of this gene was also detected in the sepal of Ranunculales (Supplementary Data Table S5 and Fig. S2). primordia, and its expression level in the carpels is constant These were from three different species of Lardizabalaceae, during the late stages (Fig. 4D–F). SIchAP3-3 expression is only one copy in each species (Supplementary Data Fig. S2). ubiquitously in the whole flower (Fig. 4G) and gradually narrows to the inner three whorls (Fig. 4H). At the late stages, SIchAP3-3 is expressed strongly in the nectar leaves, Spatiotemporal expression patterns of floral MADS-box genes but very weakly in the stamens and carpels (Fig. 4I). SIchPI in S. chinensis is expressed in the nectar leaves and stamens but not in the To detect the spatiotemporal expression patterns of floral carpels (Fig. 4J, K).

MADS-box genes in S. chinensis at the floral bud stages, we The expression patterns of A-, C- and E-class MADS-box Downloaded from performed in situ hybridization experiments. The results for genes in S. chinensis are shown in Fig. 5. SIchFL1 has very B-class MADS-box genes are presented in Fig. 4. At the strong expression signals throughout the floral organ primordia early stages, SIchAP3-1 is highly expressed in the primordia at the early stages (Fig. 5A), and high expression levels are of the nectar leaves, as well as in the androecial and gynoecial maintained in the nectar leaves, stamens and carpels at the primordia (Fig. 4A). Later on, the expression signals of late stages (Fig. 5B, C). The expression level of SIchFL2 is SIchAP3-1 were mainly detected in the nectar leaves and lower than that of SIchFL1, and its transcripts were found in http://aob.oxfordjournals.org/

ABC at Institute of Botany, CAS on August 24, 2012

DEF

GH I

JKL

F IG. 4. Expression patterns of B-class MADS-box genes of Sinofranchetia chinensis as revealed by in situ hybridization analyses: (A–C) SIchAP3-1;(D–F) SIchAP3-2;(G–I)SIchAP3-3; (J, K) SIchPI; (L) negative control with sense probe for SIchPI. (A) and (D) show a young inflorescence with multiple flowers, whereas all other panels show only one flower. Abbreviations: gp, gynoecial primordium; ap, androecial primordium; se, sepal; st, stamen; ca, carpel. Arrows indicate nectar leaves. Scale bars ¼ 100 mm. 64 Hu et al. — Expression of floral MADS-box genes in Sinofranchetia

ABC

DE F Downloaded from

GH I http://aob.oxfordjournals.org/

JKL at Institute of Botany, CAS on August 24, 2012

F IG. 5. Expression patterns of A-, C- and E-class MADS-box genes of Sinofranchetia chinensis as revealed by in situ hybridization analyses; (A–C) SIchFL1; (D–F) SIchFL2;(G–I)SIchAG; (J, K) SIchSEP3; (L) negative control with sense probe for SIchAG. Abbreviations: gp, gynoecial primordium; ap, androecial primordium; se, sepal; st, stamen; ca, carpel. Arrowheads indicate nectar leaves. Scale bars ¼ 100 mm. the floral meristems and all the floral organs (Fig. 5D–F). No expression signal of SIchAP3-2 was found in the carpels Initially, SIchAG is highly expressed throughout all the floral or leaves. In comparison, the expression of SIchAP3-3 is organ primordia (Fig. 5G). During the later stages of develop- mainly restricted in the nectar leaves in both male and ment, SIchAG is expressed in the nectar leaves, stamens and female flowers, and the expression level is higher in the carpels (Fig. 5H, I). SIchSEP3 is ubiquitously expressed in male flowers. Very low expression of SIchAP3-3 was also the whole flower, at the early and late stages (Fig. 5J, K). detected in the stamens of the male flowers, but not at a signifi- Furthermore, we investigated the expression patterns of B- cant level and not in the staminodes of the female flowers. and C-class MADS-box genes of S. chinensis in mature SIchPI is expressed at high level in the sepals, nectar leaves flowers by tissue-specific RT-PCR (Fig. 6). The mature male and stamens/staminodes in both male and female flowers and and female flowers can be distinguished from each other, at low levels in the carpels of the female flowers. In the and B- and C-class MADS-box genes are differentially female flowers, the transcripts of SIchAG were found in the expressed in the male and female flowers (Fig. 6B). staminodes and carpels. In comparison, SIchAG is expressed SIchAP3-1 is highly expressed in the nectar leaves and at relatively low levels in the nectar leaves and stamens in stamens/staminodes in both male and female flowers, the male flowers (Fig. 6B). whereas there is very low expression of SIchAP3-1 in the carpels of the female flowers. Its expression was also found in the sepals, and the expression level is higher in the Epidermal cell morphology of different floral organs female flowers than in the male flowers. In addition, a high ex- in S. chinensis pression signal of SIchAP3-1 was also observed in the leaves. There is no significant difference in morphology of the epi- SIchAP3-2 shows higher expression in the nectar leaves and dermal cells of different floral organs between female flowers stamens in the male flowers, and lower expression in the and male flowers (Fig. 7). The adaxial epidermal sepal cells sepals, nectar leaves and staminodes in the female flowers. are asymmetrically conical-papillate (Fig. 7A, I); the abaxial Hu et al. — Expression of floral MADS-box genes in Sinofranchetia 65

A B ab FS FN FSt FCa MS MN MSt LF

SIchAP3-1

SIchAP3-2 cd e f SIchAP3-3

SIchPI gh i j SIchAG Downloaded from ACTIN

F IG. 6. and floral organs with corresponding expression analyses in Sinofranchetia chinensis: (A) FAA-fixed flowers and floral organs of S. chinensis: (a) male flower; (b) female flower; (c–f) floral organs in male flower; (g–j) floral organs in female flower; (c) and (g) sepal; (d) and (h) nectar leaf; (e) stamen; (f) rudimentary carpel; (i) staminode; ( j) carpel. Scale bars ¼ 2 mm. (B) Tissue-specific RT-PCR results for SIchAP3-1, SIchAP3-2, SIchAP3-3, SIchPI, SIchAG

and ACTIN (control gene). Abbreviations: FS, female flower sepal; FN, female flower nectar leaf; FSt, female flower staminode; FCa, female flower carpel; MS, http://aob.oxfordjournals.org/ male flower sepal; MN, male flower nectar leaf; MSt, male flower stamen; LF, leaf; male flower rudimentary carpel is not included in RT-PCR. ones are flat, irregular and with slightly sunken stomata (Fig. 7E, in the floral meristems as the sepals initiated, and their expres- M). The conical epidermal cells were found on the adaxial sion domains diverged gradually during floral development, es- surface of the nectar leaf (Fig. 7B, J). The abaxial surface of pecially at mature stages (Figs 4A–I and 6B). These results the nectar leaf consists of flat and irregular cells (Fig. 7F, N). suggest that the three copies of AP3-like genes in S. chinensis The epidermal cells of the anther are irregular in shape might have undergone subfunctionalization after gene duplica- at Institute of Botany, CAS on August 24, 2012 (Fig. 7C, K), and those of the filament are flat and rectangular tion; similar cases were also reported in other Ranunculales (Fig. 7G, O). The epidermis of the carpel/rudimentary carpel species, such as Aquilegia vulgaris (Ranunculaceae) and is covered with irregularly shaped cells (Fig. 7D, H, L, P). Papaver somniferum (Papaveraceae) (Force et al., 1999; Lynch and Force, 2000; Kramer et al., 2003, 2007; Moore et al., 2005; Drea et al., 2007). DISCUSSION In addition, we compared the expression pattern of AP3-like In this study, the A-, B-, C- and E-class MADS-box genes genes in S. chinensis with that in A. vulgaris, a well-studied were found to be expressed in the nectar leaves of Ranunculaceae species. Both of these two species have three S. chinensis at different levels during different developmental copies of AP3-like genes and nectar leaves, except that the stages (Figs 4–6). It implies that these floral MADS-box genes nectar leaves in A. vulgaris have become spurs and are larger might contribute to the developmental regulation of the nectar (Kramer et al., 2007). At early stages, the expression of leaves in S. chinensis. In addition, the A- and E-class genes SIchAP3-2 and SIchAP3-3 is broader than that of corresponding display relatively broad expression patterns in most of the Aquilegia genes, AqvAP3-2 and AqvAP3-3 (Fig. 4D, G; Kramer floral organs in S. chinensis, while the B- and C-class genes et al., 2007), and SIchAP3-1 shares the same expression pattern have major expression regions, and some even show relatively with AqvAP3-1 (Fig. 4A; Kramer et al., 2007). At mature stages, specific expression in the nectar leaves at mature stages. the expression domains of SIchAP3-1, SIchAP3-2 and Therefore, the B- and C-class genes might be preferential can- SIchAP3-3 generally resemble those of AqvAP3-1, AqvAP3-2 didates to explore the nature of the nectar leaves in S. chinensis and AqvAP3-3, respectively, except for the expression of at the molecular level. SIchAP3-1 in leaves (Fig. 6B; Kramer et al., 2007). SIchAP3-3 Three AP3-like genes were identified in S. chinensis, and, im- and AqvAP3-3 are all mainly expressed in nectar leaves and portantly, SIchAP3-3 is the first representative gene of the weakly expressed in stamens but not in staminodes (Fig. 6B; AP3-III lineage from Lardizabalaceae, based on the updated Kramer et al., 2007). And another B-class gene, SIchPI, phylogenetic tree of AP3-like genes in Ranunculales (Fig. 3). which is a PI-like gene, is strongly expressed in the nectar Meanwhile, our phylogenetic analysis suggested that the leaves and stamens/staminodes at late stages, like AqvPI two major gene duplication events involved in the evolution (Figs 4K and 6B; Kramer et al., 2007). In general, the expression of AP3-like genes in Ranunculales occurred at least before pattern of B-class genes in S. chinensis and A. vulgaris is very the divergence of the Ranunculaceae, Berberidaceae, similar, especially at mature stages. More interestingly, the Menispermaceae and Lardizabalaceae (Fig. 3; Rasmussen expression pattern of B-class genes mentioned above is et al., 2009; Sharma et al., 2011). Furthermore, the three also found in other Ranunculales species with nectar leaves, AP3-like genes of S. chinensis show distinct and complex ex- such as Trollius laxus and Xanthorhiza simplicissima pression patterns (Figs 4A–I and 6B). They were all detected (Ranunculaceae), Berberis gilgiana and Epimedium grandiflora 66 Hu et al. — Expression of floral MADS-box genes in Sinofranchetia

A B C D

E F G H Downloaded from http://aob.oxfordjournals.org/

I J K L at Institute of Botany, CAS on August 24, 2012

M N O P

F IG. 7. Epidermal cell morphology of floral organs of female and male flowers in Sinofranchetia chinensis under SEM: (A–H) female floral organs; (I–P) male floral organs. (A) adaxial and (E) abaxial surface of the sepal; (B) adaxial and (F) abaxial surface of the nectar leaf; (C) anther and (G) filament of the staminode; (D) carpel of female flower; (H) surface of the carpel; (I) adaxial and (M) abaxial surface of the sepal; (J) adaxial and (N) abaxial surface of the nectar leaf; (K) anther and (O) filament of the stamen; (L) rudimentary carpel of male flower; (P) surface of the carpel. Scale bars: (A–C, E–K, M–O) ¼ 20 mm; (P) ¼ 50 mm; (L) ¼ 100 mm; (D) ¼ 250 mm.

(Berberidaceae) (Rasmussen et al., 2009). These gene- and stamens in some Ranunculales species seems more or less expression data imply that the development of the nectar corresponding to the expression in petals and stamens in core leaves in S. chinensis might be under a similar gene regulatory eudicots (e.g. Goto and Meyerowitz, 1994; Kramer et al., programme as other nectar leaves in Ranunculales species, 1998; Kramer and Irish, 1999, 2000; Lamb and Irish, 2003). It although the shapes of these nectar leaves are variable in differ- might support the reference to ‘nectar leaves’ as ‘petals’ (e.g. ent species. Taking account of the expression pattern for B-class Cronquist, 1981; Kramer et al., 2007; Kramer and Hodges, genes in other eudicots, the expression in nectar leaves 2010). Hu et al. — Expression of floral MADS-box genes in Sinofranchetia 67

What is more, the expression for the C-class gene SIchAG of share a similar genetic regulatory code with other nectar S. chinensis was observed not only in the stamens/staminodes leaves in Ranunculales species. Our study suggests that the and carpels, but also in the nectar leaves (Figs 5G–I and 6B). nectar leaves in S. chinensis could be referred to as petals, Compared with the relatively conserved and concentrated ex- and they might preserve the genetic footprint of the stamen an- pression in stamens and carpels of AG-like genes in most cestor. However, all of these need to be explored in further and angiosperms, it seems that the SIchAG gene shifts the expres- more comprehensive studies. sion domain outwards, which just fits the ‘shifting boundary’ model (Kramer et al., 2003, 2004). Furthermore, it has been suggested that the shifts in the expression domain of genes SUPPLEMENTARY DATA could lead to the genetic shifts in floral architecture and Supplementary data are available online at www.aob.oxford- result in changes in floral structure, or even homeotic transfor- journals.org and consist of the following. Table S1: primers mations (Bowman, 1997; Albert et al., 1998; Kramer et al., used for gene cloning in this study. Table S2: primers used 2003). For example, the ectopic expression of C-class genes for in situ hybridization and RT-PCR in this study. Table in the second whorl of the flower has resulted in staminoid S3: representative floral MADS-box genes used in the phylo-

petals or even true stamens instead of petals in Antirrhinum genetic analysis. Table S4: genes used in the phylogenetic ana- Downloaded from (Bradley et al., 1993). Therefore, the morphological similarity lysis of AP3-like genes in Ranunculales. Table S5: genes used between nectar leaves and stamens/staminodes in S. chinensis in the phylogenetic analysis of PI-like genes in Ranunculales. (Figs 1 and 6A) might have some relationships with the ex- Figure S1: multiple sequence alignment for AP3-like proteins pression pattern of SIchAG, which needs to be investigated of representative species from Ranunculales. Figure S2: by gene-function analysis in the future. More importantly, maximum-likelihood tree of PI-like genes in Ranunculales. the nectar leaves share more common expressed floral http://aob.oxfordjournals.org/ MADS-box genes with the stamens/staminodes than other floral organs in S. chinensis, which might reflect the close ACKNOWLEDGEMENTS genetic relationship between nectar leaves and stamens We thank Prof. Yi Ren and Dr Xiaohui Zhang for helping with (Figs 4–6). To some degree, it suggests that the nectar the collection of plant materials, Dr Liang Zhao for providing leaves in S. chinensis might be derived from stamens, which the pictures of plant materials in the field, Prof. Hongzhi Kong, is consistent with the hypothesis reported for species of Dr Wei Wang, Dr Wenheng Zhang and Prof. Pam Soltis for Ranunculaceae and Berberidaceae (Endress, 1995; Ronse De reading the early draft of the manuscript. We also thank two Craene, 2010). anonymous referees for helpful comments on the manuscript. at Institute of Botany, CAS on August 24, 2012 Based on the morphological data, there are sepals, nectar This work was supported by the National Nature Science leaves, stamens/staminodes and carpels from outer whorl to Foundation of China (grant numbers 40830209, 30530090, inner whorl in S. chinensis. Since a colourful perianth could 31100171 and 31061160184), and CAS Visiting enhance the attractiveness to potential pollinators, the showy Professorship for Senior International Scientists (2011T1S24). petaloid sepals of S. chinensis might take more responsibility for attracting potential pollinators, compared with the small greenish nectar leaves (Glover and Martin, 1998). Moreover, LITERATURE CITED the nectar leaves could secrete nectar, which could be a Albert VA, Gustaffson MGH, Di Laurenzio L. 1998. 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